1. Field of the Invention
The present invention relates to the use of a particular class of bacteriophages (“phage”) known as virulent phages that are lytic for the bacterial species Listeria monocytogenes, and which is shown in the present invention to reduce the counts of these bacteria and/or to prevent their growth in the first place, and to be useful for identifying Listeria monocytogenes, in foods products (including but not limited to the dairy industry) as well as on processing equipment and other sites in food industry facilities, and as a therapeutic agent for treating animals infected with Listeria monocytogenes. One specific example of a virulent Listeria monocytogenes phage, is a phage designated P100, recently discovered by one of the present inventors. The present invention also relates to an endolysin produced by P100 and the use of the endolysin for reducing the amount of Listeria monocytogenes in foods products as well as on processing equipment and other sites in food industry facilities, and in or on animals infected with Listeria monocytogenes and to methods that will enable additional phage that have lytic properties to be developed and/or isolated.
Phages, as antibacterial agents, have the advantage of replicating within the bacterial target. Thus, when their progeny lyse the cell and escape into the extracellular milieu, they can infect and multiply in succeeding generations of bacteria, producing progeny levels far greater than that of the binary growth of the target bacteria, thereby increasing the phage population exponentially in numbers at the expense of the bacterial targets.
The concept of using phages to identify bacterial contamination in food products {and facilities, equipment}, in general, has been described in the scientific literature (see, e.g. Greer, J. Food Prot., 49:104-109, 1986). The concept of using specific Listeria phages in particular, to identify Listeria contamination of dairy products and facilities/equipment in specific, was described as early as 1990 (Loessner et. al., Applied and Environmental Microbiology, Jun. 1990, p. 1912-1918). The present invention concern the use of a recently discovered Listeria phage with specific, essential and relevant properties, which makes it particularly suitable for identifying and controlling Listeria contamination of dairy products, facilities and equipment.
In addition to the general scientific literature on the subject, there is also patent literature that teaches the utility of phages in general to control bacterial contaminations in food processing plants and in foodstuffs. See for example U.S. Pat. No. 5,006,347 issued on Apr. 9, 1991, U.S. Pat. No. 4,851,240 issued on Jul. 25, 1989, GB 2 253 859 A published on Sep. 23, 1992 and EP 0414304A2 published on Feb. 27, 1991. However, none of the above discussed patents disclose a Listeria phage which was actually tested and shown to successfully control bacterial contamination in food processing plants and in food products. The reason for this is that all of the Listeria phages known in the art at the time of the disclosure in the previous patents were temperate phages, and were therefore not efficient at nor suitable for industrial bacterial eradication purposes. The term “temperate” refers to the fact when a strain of phage injects its DNA into a bacterial target, the phage DNA integrates into the DNA of the host cell, as a “prophage”, and can remain integrated therein for considerable periods of time. Since the prophage excises (and initiates replication and lysis) only when the host cell becomes stressed, the ensuing bacterial lysis is unpredictable and not easily controlled, which is why temperate phages do not lend themselves well to industrial applications. Temperate phages are unsuitable for industrial decontamination purposes for other reasons as well, including the fact that they can deliver unwanted and dangerous genes to the bacteria target into which their DNA integrates. In contrast, there is a class of phages that lyse bacterial targets directly, given that they do not have the molecular machinery required to integrate into the bacterial targets. Such phages are referred to as being “virulent” or “lytic” for the bacterial targets. Virulent phages against Listeria monocytogenes were discovered recently, by one of the present inventors.
The first of these virulent Listeria phages, designated A511, was described in the literature in 1990 (see Loessner et al., Applied and Environmental Microbiology, Jun. 1990, p. 1912-1918, 1990). See also DE 43 26617 C; Loessner et al., Applied and Environmental Microbiology, April 1996, vol. 62, No. 4, p. 1133-1140; and Gaeng et al., Applied and Environmental Microbiology, July 2000, vol. 66. No. 7, p. 2951-2958. The virulent phage according to the present invention belong to the Myovirdae family and have tails which contract towards the virus head. One particularly preferred phage is designated P100 and was deposited at the American Type Culture Collection, 10801 University Blvd., Manassas Va. 20110-2209 on May 23, 2002, 2002, ATCC patent deposit designation number PTA-4383.
The virulent phages described in the present invention can also be used against CFUs (colony forming units) of Listeria monocytogenes bacteria that are in biofilms, as opposed to CFUs that are planktonic. The use of temperate Listeria monocytogenes phages against Listeria biofilms has been described in the literature (see e.g. Roy et. al., Appl. Environ. Microbiol., September, 59(9):2914-7, 1993). Specifically, Roy et. al. used temperate Listeria bacteriophages H387, H387-A, and 2671 of the Siphoviridae family. While these temperate phages demonstrated some efficacy in clearing a Listeria biofilm, even when used in combination the best they could obtain was a 3.5-3.7 log reduction in counts. Roy et al indicates that such reductions “will have to be improved on to meet the recommended reduction level of 99.999% in a 30-s exposure for a chemical sanitizing agent”. As stated above, temperate phages are not predictable or readily controllable in the timing of or efficiency with which they can kill the target bacteria.
In addition to the use of the virulent phages described above, the present inventors have also found that virulent substrains can be derived from a number of temperate phage strains. These virulent substrains can be selected by techniques such as plaque isolation (in which one selects the clearest areas of a plaque, and enriches for the most virulent strains therein by repeated cycles of growing to high titer, plating for new plaques, and picking the clearest areas of the later-generation plaques). Examples of temperate phage strains, from which virulent substrains have been or will be developed, include the temperate strains designated A118, A502, A006, A500, PSA, P35, and related viruses.
The detection of Listeria is carried out in a known manner by means of procedures based on the culture of the microorganisms. The procedure described in Int. J. Food Microbiol. 4 (1987), 249-256 takes two weeks. A somewhat more rapid procedure is recommended by the International Dairy Foundation (IDF); however it takes at least 6-8 days. Because of their length, both procedures are unsuitable for a rapid identification. Both procedures are moreover labor-intensive, as for the production of individual colonies nutrient media have to be inoculated several times, and as the isolates then have to be characterized by means of biochemical and serological investigation methods.
A prerequisite for the use of immunological tests is that the antigen is expressed, which is not the case for all proteins at any time. The tests admittedly last only a few hours, but in these procedures a two-day pre-enrichment culture is needed, as the detection limit is 10-1000×103 cells. DNA probes have a detection limit of the same order of magnitude. A prior multiplication of the bacteria is therefore also necessary for these procedures: foodstuff samples or dilutions thereof are streaked out on agar plates, and the inoculated plates are incubated and then investigated in the colony hybridization procedure using a radio-labelled DNA probe. Detection is carried out by autoradiography. This method too is moreover labor and time-consuming.
The polymerase chain reaction (PCR) allows the in vitro replication of nucleic acids; a preculture is in general not necessary in this procedure. The detection limit is 0.5×103 cells. As, however, DNA from dead cells or alternatively isolated DNA is also replicated in this procedure, contamination with dead cells cannot be differentiated from contamination with living cells.
The limitations of these methods indicate the need to provide improved agents and methods for the detection of bacteria of the genus Listeria. 
EP 0 168 933 (U.S. Pat. No. 4,861,709) discloses a detection procedure for bacteria, e.g., Escherichia coli, based on the use of a recombinant bacteriophage. This phage contains the lux gene from Vibrio fischeri and thus makes possible the detection of E. coli by bioluminescence with a good detection limit (0.5×103 cells). Using temperate phages, G. J. Sarkis et al. (1995) Molecular Microbiology 15, 1055-1067 describe a detection procedure for mycobacteria. In these detection procedures, only metabolically active bacterial cells are detected; interference due to dead bacterial cells as in the PCR technique does not occur.
Scherer, et al., U.S. Pat. No. 5,824,468, issued on Oct. 20, 1998 describes a detection procedure for bacteria of the genus Listeria, where a DNA vector is prepared which includes a genetic system comprising DNA which encodes the expression of one or more detectable proteins, and a DNA vector A511 is used which specifically infects the bacteria of the genus Listeria and transfers the genetic system to the bacteria. The detectable proteins are expressed in the bacteria and detection of the detectable proteins indicates the presence of bacteria of the genus Listeria. 
Phage-encoded lysins or endolysins are highly active enzymes which hydrolyze bacterial cell walls. These phage encoded cell wall lytic enzymes are synthesized late during virus multiplication and mediate the release of progeny virions. Endolysins can be used to lyse Listeria cells to recover nucleic acids or cellular protein for detection or differentiation. The endolysin can also be used to treat animals (including humans) which are infected with Listeria and to treat surfaces which may be contaminated with Listeria. Lysins from Listeria phages (A118, A500 and A511) have been cloned and purified (Loessner, et al., Applied and Environmental Microbiology, August 1996, p. 3057-3060).
2. Description of the Related Art
Listeria monocytogenes is a bacterial pathogen that contaminates many food products, the list of which includes but is not limited to soft cheeses, patés, ice cream, smoked and cured fish, frozen seafood, salads, and processed meats. When ingested, these bacteria can produce a disease termed listeriosis, characterized by a variety of symptoms and conditions, including diarrhea, abortion, and encephalitis. Collectively, in the industrialized nations, hundreds of deaths occur each year as a result of Listeria monocytogenes food contamination.
The food processing industry has not been sufficiently successful in eradicating Listeria monocytogenes bacteria from the environment of the processing plants. As a result, even foods that have been pasteurized at temperatures high enough to kill these bacteria nevertheless become contaminated, post-pasteurization. The bacteria gain access to the foodstuffs through one or more routes, including (i) from the raw materials (e.g. raw milk, and/or milk that has been pasteurized at low temperatures); (ii) from the processing machinery (in and on which the bacteria can grow as biofilms that are difficult to eradicate ); and (iii) from airborne bacteria present in the plant environment which can settle onto the surface of the foodstuffs during curing, packaging, and so on.
Despite the numerous methods used in the food industry to control and prevent L. monocytogenes contamination, the bacteria gain access to and persist in the environment of food processing plants. Moreover, they survive the very high concentrations of salt that are present in several food-making processes. The resulting contamination of the foodstuffs (including but not limited to cheeses, pates, cold cuts, hot dogs and other processed foods) leads to scores of deaths each year in developed nations, and also to product recalls whose retail worth each year, in the aggregate, is measured in the hundreds of millions of dollars.
The methods currently in use to control Listeria in the food industry include: (i) pasteurization of primary ingredients (e.g. milk) and heat treatment of the products, which is often unsuccessful because recontamination frequently occurs and many products cannot undergo a final (listeriocidal) heat treatment; (ii) application of physicochemical agents such as disinfectants, enzymes, antibiotics, etc., which experience has shown do not reduce the bacterial counts sufficiently; and (iii) attempts to break up biofilms mechanically, which leave sufficient residues of bacteria behind that the foodstuffs still become contaminated.
Additional methods must therefore be made available to the food processing industry in order to protect the health of consumers, and to reduce the exposure of numerous companies to the great cost and the loss of good will that result from such contaminations and recalls.
The present inventors have conducted a series of experiments using a strain of L. monocytogenes that is prevalent in the processing plant of a particular manufacturer of soft (“spread”) cheeses. That bacterial strain proved to be susceptible to phage P100 in vitro. As will be shown in the Examples section, phage P100 proved able to reduce below measurable/detectable limits the Listeria bacteria that had been spiked into a cheese-like matrix.